Developmental Plasticity of Epithelial Stem Cells in Tooth and Taste Bud Renewal

Developmental plasticity of epithelial stem cells in tooth and taste bud renewal

Ryan F. Bloomquista,b,c, Teresa E. Fowlera, Zhengwen And, Tian Y. Yud, Kawther Abdilleha, Gareth J. Frasere, Paul T. Sharped, and J. Todd Streelmana,1

aSchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332; bDepartment of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA 30912; cDepartment of Restorative Sciences, Dental College of Georgia, Augusta University, Augusta, GA 30912; dCentre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London SE1 9RT, United Kingdom; and eDepartment of Biology, University of Florida, Gainesville, FL 32611

Edited by Marianne E. Bronner, California Institute of Technology, Pasadena, CA, and approved July 25, 2019 (received for review December 13, 2018) In Lake Malawi cichlids, each tooth is replaced in one-for-one enamel-secreting ameloblasts along the IEE (2, 3). Classic work fashion every ∼20 to 50 d, and taste buds (TBs) are continuously demonstrated that Sox2 marks the putative ESC niche in a cadre renewed as in mammals. These structures are colocalized in the of mammals and reptiles (4), bony fishes (5), and sharks (1, 6). + fish mouth and throat, from the point of initiation through adult- Genetic fate mapping experiments show that Sox2 ESCs con- hood. Here, we found that replacement teeth (RT) share a contin- tribute to all lineages of the dental epithelium (7, 8). uous band of epithelium with adjacent TBs and that both organs Recent work in models of stem cell-driven organ renewal (e.g., coexpress stem cell factors in subsets of label-retaining cells. We tooth, intestine, hair follicle, lung) has revealed surprising plas- used RNA-seq to characterize transcriptomes of RT germs and TB- ticity and noteworthy context dependence of epithelial cellular bearing oral epithelium. Analysis revealed differential usage of behavior (9). In each of these systems, there are multiple stem developmental pathways in RT compared to TB oral epithelia, as cell types and conditions during which plasticity between types is well as a repertoire of genome paralogues expressed complimen- favored. For example, in the intestinal crypt and the hair follicle, tarily in each organ. Notably, BMP ligands were expressed in RT cells from differentiated organ zones can regain stem cell com- but excluded from TBs. Morphant fishes bathed in a BMP chemical petence and ultimately repopulate the organ upon targeted ab- antagonist exhibited RT with abrogated shh expression in the inner lation (10). Notably, epithelial cells from outside the hair follicle dental epithelium (IDE) and ectopic expression of calb2 (a TB can migrate to the follicular stem cell niche. Once in position, marker) in these very cells. In the mouse, teeth are located on the these cells behave like endogenous stem cells (11). Likewise, in jaw margin while TBs and other oral papillae are located on the the mouse incisor, renewal is restored in certain circumstances by + − tongue. Previous study reported that tongue intermolar eminence recruitment of Sox2 cells from a Sox2 cell population (8). The (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants degree to which such heterogeneity exists in mesenchymal stem exhibited dysmorphic invagination. We used these mutants to dem- cell populations is less well studied, but consensus is emerging. onstrate altered transcriptomes and ectopic expression of dental In mouse incisor mesenchyme for instance, pericytes can be markers in tongue IE. Our results suggest that vertebrate oral epi- reprogrammed to form odontoblasts upon injury (12) whereas thelium retains inherent plasticity to form tooth and taste-like cell neural crest-derived glia (13) and Gli1-expressing periarterial types, mediated by BMP specification of progenitor cells. These find- cells associated with the neurovascular bundle (14) contribute to ings indicate underappreciated epithelial cell populations with prom- dental pulp homeostasis. Recently, we showed that Celsr1 marks a + ising potential in bioengineering and dental therapeutics. population of quiescent cells that are mobilized to replenish CD90 dental mesenchymal stem cells (MSCs), in specific response to regeneration | teeth | taste buds | stem cells incisor clipping (15). Taken together, these studies highlight the

umans have evolved long life spans but often retain organs Significance Hdamaged during their lives. This is perhaps taken to the extreme in our dentitions. One-fifth of all humans exhibit genetic Nearly one-third of adults over the age of 65 have lost all their disorders affecting teeth (primary or permanent), and nearly all teeth. We set out to understand tooth renewal in animals that humans develop dental problems (e.g., cavities) with age. Thirty have replacement and regeneration capabilities. Using cichlid percent of humans worldwide over the age of 65 have none of fishes and mouse models, we discovered plasticity between their 32 natural teeth remaining in their mouths (World Health tooth and taste bud progenitor cell derivatives, mediated by Organization). Whereas nonmammalian teeth are replaced de BMP. Our results suggest that oral organs have surprising re- novo throughout life via various mechanisms (1), mammals have generative capabilities and can be manipulated to express largely lost this dental regenerative ability. For example, hu- characteristics of different tissue types. mans replace each tooth only once, and mice never replace their teeth. Instead, mice and other rodents exhibit continu- Author contributions: R.F.B., P.T.S., and J.T.S. designed research; R.F.B., T.E.F., Z.A., T.Y.Y., ously growing incisors wherein enamel is renewed asymmetri- and K.A. performed research; R.F.B., T.E.F., Z.A., T.Y.Y., K.A., G.J.F., P.T.S., and J.T.S. ana- cally on the labial (outside) surface, which bears the brunt of lyzed data; and R.F.B., G.J.F., P.T.S., and J.T.S. wrote the paper. primary mastication. The authors declare no conflict of interest. In the mouse, the base of each incisor contains a region called This article is a PNAS Direct Submission. the cervical loop (CL), the location of an epithelial stem cell This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). (ESC) niche that has become a powerful model for under- Data deposition: The data reported in this paper have been deposited in the Gene Ex- standing stem cell (SC) biology. In the incisor CL, a histologically pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. distinct group of mesenchymal-like epithelial cells called stellate GSE122501, GSE128942, and GSM3688726–GSM3688731). reticulum (SR) lie sandwiched in between the inner enamel 1To whom correspondence may be addressed. Email: [email protected]. epithelium (IEE) and outer enamel epithelium (OEE). A subset This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of cells from within the SR serve as ESCs, differentiating into 1073/pnas.1821202116/-/DCSupplemental. transit amplifying (TA) cells that will multiply to generate Published online August 19, 2019.

17858–17866 | PNAS | September 3, 2019 | vol. 116 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1821202116 Downloaded by guest on September 28, 2021 importance of the niche-signaling environment, which can impinge characteristics between integument-derived epithelial appendages, upon internal molecular programs to mediate reversible behavior including the growth of hair from teeth (21) and the transition of epithelial and mesenchymal stem cells (16) and/or mobilize from scales to feathers (22). In the latter case, transcriptome populations of cells otherwise quiescent. Heterogeneity and plas- analyses implicated specific pathways, β-catenin and retinoic ticity are fundamental features of stem cell systems that must be acid, in the process (23). understood as bioengineers and developmental biologists seek to Given the integration of teeth and taste buds during cichlid manipulate cell biology for regenerative therapies. early development (17), we were prompted to investigate the Developmental plasticity is also apparent between organ sys- renewal and regeneration of these organs at later stages. To this tems. In previous work, we showed that developing teeth and end, we 1) identified label-retaining cells (LRCs) using a pulse– taste buds in cichlid fishes share a bipotent epithelium during chase strategy and 2) examined the activity of adult stem cell early patterning stages, from which dental and taste fields are markers in these adjoining structures undergoing lifelong re- specified (17). Small molecule manipulation of the Wnt, Hh, and newal (taste buds) and replacement (teeth). We performed un- BMP pathways, during the critical developmental window when biased RNA-seq of replacement tooth and taste bud tissues and organ fields differentiate one from the other, provides clues to highlighted the role of BMP ligands in replacement teeth (RT) the regulatory logic of cichlid tooth and taste bud copatterning. and not taste buds (TBs). Finally, in both fish teeth and on Wnt signaling couples tooth and taste bud density and acts up- mouse tongues, we manipulated the BMP signaling environment stream of Hh and BMP, which together mediate organ plasticity. and demonstrated striking interorgan epithelial plasticity between Notably, treatment with the small molecule LDN, which inhibits teeth and taste-like papillae. BMP signaling, results in a striking phenotype wherein tooth density is reduced and taste buds infiltrate the tooth field. These results Results resemble studies of the integument wherein BMP-modulated Expression of Putative Stem Markers Reveals a Highly Potent Oral transgenic K14-Noggin mice demonstrated “trans-differentiation” Epithelium Connecting Teeth and Taste Buds. We began our study of sweat glands from the distal limbs into hairs (18), and both with the aim of identifying the spatial location of putative stem spatial and temporal modulation of BMP through development cell niches in both renewing TBs and successional teeth through and lentivirus manipulation specified the fate of sweat glands versus in situ hybridization (ISH). Three distinct stages of cichlid replace- hair in mice (19). ment tooth maturation are known: initiation, which encompasses

The relationship between teeth and taste bud renewal has recently placode or successional dental lamina stages of odontogenesis; BIOLOGY + “ been established in the shark wherein a sox2 odontogustatory differentiation, which encompasses cap and bell stages; and se- DEVELOPMENTAL band” (OGB) has been demonstrated to share a progenitor pool cretion, which encompasses late bell and mineralization stages (5). of cells associated with taste buds that migrate into the dental We chose to focus our study on the latter because RT spend the successional lamina (6). Similar results have now been observed longest time in this stage and secretion is the most consistent in during the development of the beaked dentition in pufferfish histology across different aged cichlids. We conducted our ISH (20). Additionally, recent study demonstrates the conversion of experiments on cichlid fry (∼30 days postfertilization [dpf]),

Fig. 1. ISH identification of tissue in RT-TB unit. Expression of pitx2 in cichlid RT germ and oral epithelium (A), calb2 expressed in TB (B), and foxa2 across RT and TB epithelium (C) during the secretion stage of cichlid replacement tooth development. Functional tooth is outlined in magenta. Shown are vibratome sections in the sagittal plane at 15-μm thickness, imaged at 40× magnification. Labial is oriented to the bottom and oral toward the left of the page. Schematic (D) shows tissue architecture with taste bud in light blue, epithelium in purple, erupted functional tooth in magenta, replacement tooth mesenchyme in pink, and ossified tissue in green. Time scale of experimental design for pulse–chase experiments (E) with 0, 4, 20, 40, and 100 days chase (DC) on top and black arrowheads representing tooth generations below. Pulse (BrdU incorporation) was started at either 4 or 20 dpf, and killing was made at either 40 or 100 DC. Many cells labeled by IHC for BrdU after 40 DC (F) with discrete cell populations by 100 DC (G, and schema in H). When pulsed beginning at 20 dpf, again followed by a 100 DC (I), fewer cells are labeled, with discrete populations still apparent (J). Labial oriented to the bottom and oral to the left of the page. Paraffin sections in sagittal plane at 15-mm thickness, imaged at 40× magnification. Epithelium outlined in black (A–C) or white (F, G, I) with taste buds outlined in circles.

Bloomquist et al. PNAS | September 3, 2019 | vol. 116 | no. 36 | 17859 Downloaded by guest on September 28, 2021 Fig. 2. ISH of adult proliferation and stem markers. Expression of trp63 (A), sox2 (B), bmi1 (C), lgr4 (D), igfbp5 (E), hopx (F), gli1 (G), celsr1 (H), and sox10 (I) during the secretion stage of cichlid replacement tooth development. Functional tooth is outlined in magenta. Shown are vibratome sections in the sagittal plane at 15-μm thickness, imaged at 40× magnification. Labial is oriented to the bottom and oral toward the left of the page. Epithelium outlined in black dashed lines and functional teeth in magenta, with taste buds outlined in circles.

developing their third generation of teeth. We first used pitx2,a adult stem cells in a host of organs (33), is essential for incisor marker of dental tissues in vertebrates, including cichlids (24) and renewal through the repression of Ink4a/Arf and Hox genes (34) + mice (25), to identify those cells belonging to the RT germ but while, in TBs, it appears that a population of Bmi1 SCs renew noted expression along the successional dental lamina, along keratinized epithelial cells distinct from SCs that renew TB cells basement membrane cells deep in the oral epithelium, both labial themselves (35). We observed expression of bmi1 distinct from and lingual to the taste bud unit (Fig. 1 A and D, schematic). As that of sox2, diffusely across TBs and surrounding epithelium we have done before in cichlids (17, 26) and has been done in and in RT epithelium. The difference between sox2 and bmi1 trout (27), we used calretinin (calb2) to mark TBs, which more expression is not surprising. In the mouse intestine, Bmi1 marks specifically was expressed in the elongated taste bud intragemmal a population of stem cells that is relatively quiescent and acti- cells and the support perigemmal cells that surround it (Fig. 1 B vated in response to injury while another distinct population of and D, schematic). We attempted to further characterize the taste Lgr5-positive stem cells are more active and responsible for bud unit using foxa2, a marker of the endoderm and taste buds in regular renewal of the crypt unit (36), and, further, each pop- mice (28) and in zebrafish (29), but, to our surprise, foxa2 not only ulation responds differently to tissue perturbations, such as ap- marked TBs but was strongly expressed in all RT cells and across optosis (37). While no homolog of Lgr5 exists in teleost fishes, outer oral epithelium associated with these two organs (Fig. 1C). lgr4 was expressed in cichlid RT and TBs more similarly to that We turned our focus to markers of, or associated with, adult of sox2, although more restricted to the basal layers of epithe- stem cells. trp63 is a p53 transcription factor family member and lium in and around the taste unit. This is an intriguing result, a well-known marker of proliferation and mitotic activity. Trp63- + + given that Lgr5 cells replenish Sox2 cells after genetic deletion deficient mice exhibit both anodontia and a thin degenerate of Sox2 in mouse incisors (8). igfbp5, shown coincident in ex- tongue epithelial layer (30). A hypothesized bipotent progenitor pression to lgr5 in gecko RT dental lamina (38), indeed colabels layer shared between filliform and fungiform taste papilla is – marked by Trp63 in mice (31). We found expression of trp63 to the basal epithelial cells associated with the regenerating RT TB mirror expression of the progenitor layer in mice (Fig. 2A), being unit. Meanwhile, Hopx, which has been used to label SCs in intestine (39) and hair follicles (40), is expressed in the sox2/lgr4/ strongly expressed in the basal cells surrounding the intragemmal + + TB cells continuing across the oral epithelium overlying the igfbp5 and bmi1 populations, as well as strongly within the dental lamina and in the CLs of the RT epithelium, in a pattern dental lamina. We noted expression of bmi1, igfbp5, and hopx in complimentary to that of sox2. Sox2 is one of the most studied mesenchyme, as well as in epithelium (Fig. 2). Markers of mes- factors in taste bud development, stem cell biology, and, more enchymal stem cells (MSCs) were indeed expressed in oral recently, in tooth regeneration. While Sox2 is a well-known TB mesenchyme, but also across epithelial tissue. gli1 was expressed marker, important for both the generation of TBs as well as across dental and TB mesenchyme, as well as in dental epithe- maintenance of TB stem cell populations (32), it has more re- lium, but absent from taste and surrounding oral epithelium (Fig. cently been implicated in dental ESCs of both mice (7) and other 2G). celsr1, recently described as a marker of quiescent cells in vertebrates (4). It has also been demonstrated as a marker of mouse dental mesenchyme (15), and sox10 were expressed in the shared progenitor cells in the shark OGB, giving rise to cells in epithelium of RT, the mesenchyme subjacent to epithelium in the tooth/taste bud interface (6). We found sox2 expression in the TB and dental lamina, and outside of the dental papilla itself TBs, as well as RT and across epithelium associated with the two but in crypt mesenchyme near the CLs (Fig. 2 H and I). Taken structures (Fig. 2B). Bmi1, a Polycomb group gene required for together, our ISH data indicate that markers of adult ESCs and

17860 | www.pnas.org/cgi/doi/10.1073/pnas.1821202116 Bloomquist et al. Downloaded by guest on September 28, 2021 MSCs are expressed within RT, TBs and the dental successional dental epithelial (ODE) cells, analogous to mammalian OEE, lamina connecting the two organs. and associated with the CL, while LRCs were located in a subset of cells closer to the inner dental epithelium (IDE) and the apex Colabeling with Stem Markers and Nucleoside Chase Identifies Stem of the dental papilla (Fig. 3 A and C). The lack of overlap be- Cell Niches across the Oral Epithelium. To more precisely identify tween these markers after a chase period is indicative of a dis- SC niche environments in cichlid RT and TBs, we conducted tinction between putative TA cells, labeled by Trp63, and putative nucleoside pulse–chase experiments, one of the primary experi- quiescent populations, labeled by BrdU. In contrast, Sox2 was ments done to first identify stem cells in the mouse incisor (41). found coexpressed with LRCs associated at the base of taste buds By exposing animals to the synthetic nucleosides 5-bromo-2′- in perigemmal cells (Fig. 3D) and at the tips of RT (Fig. 3E). deoxyuridine (BrdU) or 5-chloro-2′-deoxyuridine (CldU), it is Bmi1 protein colabeled a smaller subset of LRCs; its domain in incorporated into newly created cells, and, once removed, only the epithelium of both organs was largely in the more superficial those cells that are slow cycling or nondividing, a property of cells, many of which were negative for label retention (Fig. 3 F and stem cells, will be label-retaining cells (LRCs). We bathed cichlid G). Finally, β-cat, a regulator of stem cell maintenance and dif- fry in a solution containing BrdU at pharyngula stage (4 dpf) for ferentiation through Wnt signaling (43, 44), was coexpressed with a period of 1 wk and then killed sequentially until LRCs were epithelial and mesenchymal cells after pulse at 20 dpf and no identified (Fig. 1E). Numerous cells retained the BrdU label chase period and after pulse at 4 dpf and 100-d chase. β-cat was after 40 d of chase, but, by 100 d (at least 4 cycles of replacement active in LRCs not marked by Sox2, at the intragemmal tip of the teeth), discrete cell populations were apparent (Fig. 1 F and G). taste bud and at the cervical loops and papilla of the RT (SI By 100 d of chase, there was a high density of LRCs across all Appendix, Fig. S1 and Fig. 3 H and I). Taken together, we ob- + + epithelium labial to the FT, within the TB unit and within the served distinct populations of LRCs: Sox2 /LRC populations at + RT. We also detected LRCs in the mesenchyme, mostly associ- the base of taste buds, in the tips of teeth, and in the CLs; β-cat / + ated in a band approximate to the epithelium and in the dental LRC populations at the base of teeth and in the tips of taste + papilla. In order to compare the effects of early versus late pulse, buds; and distinct Trp63 and LRC populations following a chase we repeated these experiments beginning with a pulse beginning period. Colabeling of Sox2/β-cat/Bmi with LRCs confirmed the at 20 dpf, again followed by a 100-d chase period. We found that presence of an SC-rich epithelium, and, within it, distinct SC both the late pulse and long chase groups resulted in fewer LRCs niches became apparent: those associated with the base of the (Fig. 1I), likely because many founder stem cells are formed taste bud analogous to murine TB SCs (32), those associated with BIOLOGY

early in development (42). the incisor CL (7, 13), and an SC niche not described in the dental DEVELOPMENTAL Because ISH revealed the expression of multiple adult stem literature, occupying cells at the tip of the maturing RT. This niche markers within RT and TBs, we used double labeling of BrdU has been understudied likely because very little to date has been LRCs with immunohistochemistry (IHC) to better characterize published on the stem cell populations involved in whole tooth putative stem niches associated with these organs. In accordance replacement in a one-for-one replacement system. with mRNA expression, Trp63 protein, a marker of proliferation, was detected in TB support cells and coexpressed with RNA-Seq Reveals Unique Transcriptomes of Replacement Teeth and LRCs across the basal cells of outer oral epithelium (Fig. 3 A and Taste Buds. Because cichlid teeth and taste buds are copatterned B). After 20-d pulse and immediate sacrifice, (SI Appendix,Fig.S1), during early development and because we know little about the Trp63, a marker of proliferation, and BrdU cells were over- molecular biology of vertebrate tooth replacement, we used an lapping at the tip of the tooth, marking proliferating cells that unbiased RNA-seq approach to characterize gene expression are likely undergoing transient amplification (TA). After 4-d profiles in adult cichlid replacement teeth and taste buds. We pulse and subsequent chase, Trp63 was detected in outer dissected a band of epithelium, just labial to the outer row of

Fig. 3. Double label for LRC and IHC of adult proliferation and stem markers. Fluorescent labeling after BrdU pulse at 4 dpf and killing at 100 days chase with BrdU labeled in red and protein markers in green. Yellow/orange indicates coexpressing cells. Functional tooth is outlined by solid line, epitheliumoraland taste epithelium by dashed lines. Trp63 (A) is a marker of proliferation; labial is oriented to the left and oral toward the top of the page. A zoom-in of taste buds (B, Trp63; D, Sox2; F, Bmi1; H, B-cat) and teeth (C, Trp63; E, Sox2; G, Bmi1; and I, B-cat) shows zones of proliferation, label retention, and stem marker expression, with labial oriented to the bottom and oral to the left of the page. Shown are paraffin sections in the sagittal plane at 15-μm thickness, imaged at 40× magnification.

Bloomquist et al. PNAS | September 3, 2019 | vol. 116 | no. 36 | 17861 Downloaded by guest on September 28, 2021 Fig. 4. RNA-seq of RT and TB bearing epithelium. Heat map (A) of differentially expressed genes shows clustering based on sample type (x axis; with species data) and log fold change (heat; yellow to blue) for individual genes (y axis). ISH expression bias in TBs bearing epithelium chl1 (B), gad1 (C), klf4 (D), and sox14 (E), or in RT tissues fgf10 (F), sema3e (G), shox2 (H), and sp7 (I) for genes significantly differentially expressed in sequencing analysis. Shown are vibratome sections in the sagittal plane at 15-μm thickness, imaged at 40× magnification. Labial is oriented to the bottom and oral toward the left of the page. Tiss., tissue; Sp., species; DEG, deferentially expressed genes.

adult cichlid functional teeth, which contained a high density of temporal effects in development (19). Given differential ex- taste buds (17). We then removed the periosteum surrounding pression of BMPs, functional enrichment of this pathway in the dental bony crypts and isolated replacement tooth germs, at cichlid replacement teeth, and previous demonstration that BMP secretion stage, easily identified by their hypermineralized inhibition results in ectopic taste bud formation in dental field acrodin cap. We pooled taste bud-bearing epithelial tissues and progenitor epithelium (17), we further explored the spatial ex- replacement tooth germs for each animal, extracted RNA, pre- pression of BMP ligands in cichlid replacement teeth. We ob- pared RNA libraries, and performed RNA-seq on the Illumina served that bmp2 and bmp4 were sharply expressed in replacement 2500 platform. High quality reads were aligned to the Malawi cichlid reference genome (on average, across all samples, over tooth epithelium and mesenchyme but excluded from all tissues in 95% of reads mapped to the reference). Fragment counts across and around the taste bud (Fig. 5 A and B). In turn, we bathed all samples were obtained, normalized, and fit to a linear model cichlids in the small molecule inhibitor of BMP, LDN, for a period to determine differential expression between tissue types. Genes were considered significantly differentially expressed between teeth and taste buds if they exhibited both a 2-fold expression difference or greater and an adjusted P value of <0.05. Using this criterion, we found that 3,902 genes were differentially expressed between the tissue types (Fig. 4). Of those, 2,482 were up- regulated in dental tissues while 1,420 were up-regulated in taste buds (Dataset S1). Significant tooth-biased genes included axin2, bmp2, bmb4, bmp6, bmp10, dlx4, dlx5, dlx6, edar, fgf10, msx2, pax9, pitx2, runx2, sostdc1, and wnt7b. Genes biased in taste buds included avpr2, barx2, dmbx1, egfr, osr1, six3, and sox14. For a subset of these genes, we confirmed differential expression by ISH (Fig. 4 C–I). Notably, we identified hundreds of genome paralogues expressed complementarily in RT or TBs, respectively. Paralogues included glutamate receptors, keratins, solute carriers, andzincfingerproteins(Dataset S2). Given the role of paralogous genes in evolutionary novelty (45), we were intrigued by this example of complementary paralogue expression in adjacent oral organs. Six of 15 loci implicated by GWAS in human tooth number (ajuba, bmp4, calu, cacnb2, cdon,andvcl)(46)were expressed in cichlid replacement teeth. We used GeneAnalytics (47) and ToppGene (48) to identify pathway and phenotype enrichment based on lists of differentially expressed genes (Dataset S3). Notable statistical enrichment in replacement teeth included the categories “odontogenesis,”“bone morphogenesis,”“focal adhesion,”“mesenchymal stem cell differentiation,”“Wnt signaling,”“TGF-beta signaling,”“extracellular matrix organization,” and “abnormal molar morphology.” Categories enriched in taste buds included “epidermis development,”“abnormal tongue epithelium morphology,” and “signaling by ERBB2.”

Manipulation of the BMP Pathway Uncovers Interorgan Plasticity Fig. 5. BMP expression and effect of LDN on RT SC differentiation. bmp2/4 bias to RT (A and B). Solvent control RT express shh (C) and not calb2 (E)inCL between RT and TBs during Regeneration and Renewal. The pleio- epithelium, but LDN-treated RT express calb2 (F) and not shh (D). Shown are tropic effects of BMPs on multiple epithelial appendages has vibratome sections in the sagittal plane at 15-μm thickness, imaged at 40× been well established, such as Bmpr1a control of both tooth and magnification. Labial is oriented to the bottom and oral toward the left of hair differentiation (49), and the role of BMPs in integument the page. Epithelium outlined in black dashed lines and functional teeth in transdifferentiation has been demonstrated to have spatial and magenta, with taste buds outlined in circles.

17862 | www.pnas.org/cgi/doi/10.1073/pnas.1821202116 Bloomquist et al. Downloaded by guest on September 28, 2021 BIOLOGY DEVELOPMENTAL

Fig. 6. Mouse tongues lacking Follistatin misexpress dental markers in the intermolar eminence. Dorsal view of whole mount ISH for a panel of tooth − − markers shows misexpression of these genes in Follistatin mutants (Fst / ). Quantification of this effect by qRT-PCR follows dissection of intermolar eminence tissue. (Magnification: 10×.)

of 48 h and then killed to characterize treated replacement teeth we were intrigued by results from Beites et al. (51), who dem- − − at the cellular and transcript level. Shh regulates preameloblasts in onstrated that mouse tongues in Follistatin mutants (Fst / ) the rodent incisor transitioning from stem cell-derived TACs to exhibited dysmorphic epithelium in the posterior intermolar − − enamel-secreting ameloblasts (50). In control (dimethyl sulfoxide eminence (IE). The IE epithelium of Fst / mice invaginates and [DMSO]) animals, shh was expressed in the analogous inner expresses Sox2, Shh,andFoxa2 (51), similar to cichlid teeth (ref. dental epithelium of cichlid teeth (Fig. 5C). Upon exposure to 17 and above), but somewhat distinct from more anterior gustatory LDN, shh expression was undetectable, and replacement teeth mouse tongue papillae. Thus, we first examined the transcriptomes appeared shorter and malformed, particularly in the cervical of dissected IE epithelium from embryonic day 17.5 (E17.5) − − loop regions (Fig. 5D). We then assayed expression of the taste wild-type and Fst / littermates. This unbiased approach identified bud marker calb2 in LDN-treated animals and observed expression ∼700 differentially expressed genes (Dataset S4). We hypothe- of calb2 in cervical loop epithelium in regions where shh expression sized that those genes up-regulated in this context might carry a was abrogated (Fig. 5F). These data suggest that temporary loss of tooth-like signature. Indeed, this gene set was enriched for bi- BMP signaling in cichlid replacement teeth results in dental cells “ adopting taste bud characteristics. ological process Gene Ontology (GO) categories bone morphogenesis,”“Wnt signaling,” and “odontogenesis” (Dataset Tongue Papillae of Fst−/− Mice Adopt the Characteristics of Teeth. S3). Notably, the overlap in GO biological process terms be- − − Given the protooth role of the BMP pathway in our data and tween genes up-regulated in Fst / IE papillae and those elsewhere, as well as the expression of BMP antagonists (fst, enriched in cichlid replacement teeth is statistically significant − sostdc1, and osr2) in embryonic taste territories in cichlids (17), (Fisher’s exact test, P < 2.2 27; shown graphically in SI Appendix,

Bloomquist et al. PNAS | September 3, 2019 | vol. 116 | no. 36 | 17863 Downloaded by guest on September 28, 2021 where label-retaining cells reside: 1) labial and 2) lingual cervical loops (CLs) (similar to mouse teeth), 3) at the tip of the tooth, 4) dental pulp mesenchyme, where the tooth is innervated by a neurovascular bundle (NVB), and 5) surrounding the taste bud unit. Stem cell factors Bmi1 and Sox2 colabel subsets of LRCs in the epithelium of replacement teeth and taste buds (Figs. 2 and 3). The tooth tip niche has not been observed in other systems and is particularly interesting. The tip of the replacement tooth is located in close proximity to the dental successional lamina and likely acts as a signaling center to direct tooth morphogenesis (53) (Fig. 7). As we have demonstrated previously for early tooth and taste bud development (17), these organs share syn-expression of many factors during regenerations stages, but BMP ligands ap- pear confined to the tooth zone. Knockdown of BMP signaling results in a striking phenotype wherein dental epithelium expresses calb2, a marker of taste bud fate (Fig. 5). This effect is likely due to the loss of BMP activity in the dental zone such that cells migrating to the dental successional lamina (e.g., ref. 6) or cells already present in the replacement tooth CLs are transfated (Fig. 7). We used mice null for the BMP antagonist Follistatin (Fst) to examine predictions associated with increased BMP signaling on the tongue (51). Notably, the intermolar eminence − − tongue epithelium of Fst / mice invaginates (51), exhibits a Fig. 7. A model of stem cell niche localization and associated stages of modified tooth-like transcriptome, and expresses classic dental tooth regeneration in the cichlid. Schemes are based on a sagittal section markers like Dlx1, Msx1, Msx2, Pax9, and Pitx2 (Fig. 6 and SI through a single functional tooth and its replacement within the lower jaw. – (A) During maturation of the replacement tooth, a number of potential stem Appendix, Figs. S2 S4). The data in mouse model suggest latent cell pockets are identified (yellow). These include regions associated with (i) plasticity among teeth and tongue papillae, even when the organs taste buds in the oral epithelium, (ii) at the tip of the tooth in association are not colocalized or linked via a successional lamina. with the enamel knot-like structure, and (iii) in the cervical loop-like regions The last decades of research in numerous organ systems have of inner dental epithelium of the developing tooth. (B) As tooth regenera- uncovered significant intraorgan plasticity of epithelial cells in tion initiates, an epithelial invagination is triggered by reciprocal interac- tissue regeneration (9). Examples of interorgan plasticity are tions of epithelial stem/progenitor cell compartments and the underlying perhaps even more dramatic. Such examples include the case of neural crest mesenchyme where stem cells are also thought to be active (at conditional deletion of Med1 in the mouse incisor, which the onset of dental lamina invagination: green; and surrounding the pro- liferative progression (red arrows) of the successional lamina (red star) to- switches dental to epidermal fate, such that hairs grow in the ward tooth regeneration: pink). (C) During the differentiation phase of place of teeth from renewing postnatal dental epithelia (21). successor tooth development, signals emanate from the surrounding mes- Similarly, Wu et al. showed how multiple pathways could each enchymal papilla and dental follicle (green). Signals from the oral epithelial partly convert scales to feathers (22). We demonstrate a change compartments associated with taste territories are still directing tooth devel- in interorgan characteristics shown consistently in fishes and opment and growth (red arrows). (D) As the successor tooth matures and is mouse. Two general concepts are central to these transforma- ready to replace the functional tooth, signals from the epithelium and the tions (SI Appendix, Fig. S5) in either developing or renewing dental follicle (pink) start to form the support tissues. As the tooth makes its organs, as well as the more common cases of intraorgan plas- way toward functionality, signals from the EK-like structure (green star) may ticity. First is the idea of a ground state for cells (committed or direct the epithelial stem/progenitor niche associated with the oral taste ter- ritory (red arrow) and the underlying mesenchyme (green) to activate further multipotent) in tissues that develop from common epithelium. tooth regeneration as the successor tooth tip (green star) moves past the site In the examples explored above, dental fate is layered upon a of the oral epithelial niche (green arrow). Epithelium outlined in black dashed sensory (taste bud) or epidermal ground state. Manipulation of lines, with taste buds outlined in dashed lined circles (red labial, black lingual). the niche signaling environment can coax precursor cells back into a ground state where differentiation into different placode derivatives can occur (SI Appendix, Fig. S5). Second is the clear Fig. S2). Next, we examined the spatial domain of tooth markers spatial and temporal context dependency of the niche-signaling up-regulated on Fst-null tongues. Using in situ hybridization and environment. Better understanding of the evolutionary and de- qPCR, we confirmed that numerous dental markers are misex- velopmental lineages of cells that function in oral organ systems (54) pressed in the IE of mouse tongues lacking Follistatin. These in- may provide clues to their manipulation in regenerative medicine. clude Bmp7, Shh,andFoxa2 (as shown by ref. 51), as well as Gli1, Osr2, Msx1, Msx2, Pax9, Pitx2, Barx1,andDlx1 (Fig. 6 and SI Ap- Materials and Methods pendix,Figs.S3andS4). Our experiments in mouse mirror those Cichlid Husbandry. Adult Malawi cichlids were housed in recirculating conducted in cichlids and highlight a surprising long-term plasticity aquarium systems at 28 °C (Georgia Institute of Technology) for embryo production. Species of Lake Malawi cichlids included Labeotropheus fuele- between dental and other oral organ types, mediated by BMP. borni (LF), Metriaclima zebra (MZ), Petrotilapia chitimba (PC) and were se- lected based on embryo availability, with a preference toward MZ, owing to Discussion their genome assemblage (55) and partial albinism morph, which permitted Here, we report three main discoveries. Cichlid one-for-one, better imaging of histological stain. Fertilized embryos were harvested from cycled tooth replacement occurs in anatomical linkage with taste mouth-brooding females and staged in dpf according to Nile Tilapia de- – buds undergoing continuous renewal. These two structures, teeth velopmental series (56). Embryos were raised to desired stages for ISH, pulse chase experiments, or chemical treatment and euthanized with buffered MS-222 for and taste buds, are colocalized in the oro-pharynx of most fixation in either 4% paraformaldehyde or 10% neutral buffered formalin. nonmammalian vertebrates, and the two organ types share developmental precursors and deep molecular homology (17, 52). Cichlid In Situ Hybridization. Primers for target probe sequence were designed We identified 5 anatomical zones (Fig. 7), or stem cell niches, using the published and annotated genomes of tilapia species Oreochromis

17864 | www.pnas.org/cgi/doi/10.1073/pnas.1821202116 Bloomquist et al. Downloaded by guest on September 28, 2021 niloticus (55) and the aligned genome of Malawi cichlid M. zebra from the standard chloroform extraction, RNeasy mini columns (Qiagen) were utilized University of Maryland Cichlid Blast Server Tool. It has been reported that to purify RNA for storage at −80 °C. Total RNA was quantified using Qubit genomic sequence diversity across the Lake Malawi assemblage is 0.28%, less (Molecular Probes) and quality analyzed using the Agilent 2100 Bioanalyzer than reported values for laboratory strains of zebrafish (57), and riboprobes System for RNA library preparation. RNA input was normalized to 1 μg, and were reactive across Malawi cichlid species. Target sequences were transformed libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit and cloned, and sequences were deposited in GenBank (26). Riboprobes were (Illumina-Kit A). Libraries were again quantified, quality assessed, and nor- synthesized and labeled with Digoxigenin (DIG) (Roche) using the Promega malized for sequencing on the HiSeq 2500 Illumina Sequencing System. System Sp6/T7. In situ hybridization was performed using previously published methods in whole mount (24) and visualized using an alkaline phosphatase Cichlid Transcriptome Analyses. Raw sequence reads from RT and TB samples (AP)-conjugated anti-digoxigenin antibody (Roche) to activate an NBT/BCIP were quality controlled using the NGS QC Toolkit (58). Raw reads with an (Roche) blue color reaction. Specimens were embedded in chick albumin and average Phred quality score below 20 were filtered out. The remaining reads cross-fixed with 2.5% glutaraldehyde followed by being postfixed with 4% were further trimmed of low-quality bases at the 3′ end. Quality-controlled μ paraformaldehyde (PFA). Histological sections were cut at 18 to 20 musinga reads for each sample were aligned to a recently improved M. zebra Leica Microsystems VT1000 vibratome and then mounted with glycerine for reference genome (59) using TopHat v2.0.9 (60). The resulting TopHat2 output × × imaging using a Leica DM2500 compound microscope with 20 to 40 objectives. bam files were sorted and converted to sam files using samtools v0.19 (61). Sorted sam files were used as input for the HTSeq-count v0.6.1 program to BrdU/CldU Labeling. BrdU pulse–chase experiments were carried out to label obtain fragment counts for each locus (62). Fragment counts were scale- slow-cycling cells, a property of stem cells. Specimens reared to 4 dpf were normalized across all samples using the calcNormFactors function in the bathed in either a 2% solution of BrdU in vivo labeling reagent (00-0103; edgeR package v3.6.8 (63). Relative consistency among replicates and samples Invitrogen) or in 200 mL of fish room water at 28 °C in an Erlenmeyer flask. A was determined via the Multidimensional scaling (MDS) feature within the similar pulse was performed on fish reared to 20 dpf using a 0.1 molar stock edgeR package in R. Scale-normalized fragment counts were converted into solution of CldU (C6891; Sigma) made with DMSO. This solution was added log2 counts per million reads mapped (cpm), with precision weights using to 200 mL of fish water in 100-mL aliquots three times daily for a period of voom, and fit to a linear model using the limma package v3.20.9 (64, 65). 1 wk. Daily, 1-mL aliquots of BrdU/CldU solution were added for a total la- Pairwise contrasts were constructed between RT and TB samples. After cor- “ ” × beling period of 1 wk to complete the pulse period. Embryos were rinsed 2 recting for multiple comparisons using the Benjamini–Hochberg method (66), and then moved to fresh water at 28 °C in a recirculating aquarium system genes were considered differentially expressed between RT and TB samples if (GIT). Embryos were killed over 20-d periods up until a period of 100-d “chase.” they exhibited both a fold change ≥2andPadj < 0.05. Data have been de- This period was verified by immunohistochemistry as the chase time point posited in NCBI GEO under accession code GSE122501. where only discreet populations of slow-cycling cells were labeled. Functional Overrepresentation Analyses. Functional enrichment of differen- BIOLOGY

Immunohistochemistry. Embryos were killed as described and fixed in 10% tially expressed genes was identified using the comprehensive Gene Analytics DEVELOPMENTAL NBF at room temperature at 4 °C. Embryos were then rinsed in phosphate- (47) and ToppGene (48) tools. buffered saline (PBS) and decalcified for a period of 48 to 72 h in a mild acid (0.1 M EDTA) at room temperature before being processed through a graded Mouse Husbandry. All mouse work was performed according to Home office series of EtOH (25%, 50%, 75%, 100%, 100%) and 2 washes in xylene. Em- guidelines in the United Kingdom and approved by the King’s College bryos were washed in xylene for 3 h and incubated 60 °C and embedded in + − London animal ethics committee. Follistatin heterozygotes mice (Fst / ) paraffin for sectioning on a Thermo Scientific Microm HM355S microtome at were from JAX (stock no. 002788), and C57BL/6 mice were from CRL (Charles 5 μm. Slides were dried for 24 h at 42 °C and rehydrated through xylene and a + − River Laboratory). Fst / mutant mice were bred with C57BL/6 for mouse graded series of EtOH for incubation in blocking solution (3% goat serum, 1% strain maintenance because homozygous mice die immediately after birth. bovine serum, 0.1% Triton X-100) for 1 h at room temperature. Slides were then incubated overnight in a 1:100 dilution of anti-rabbit primary antibody (rabbit anti–β-cat [GTX26302; Genetex], rabbit anti-Sox2 [GTX124477; Gene- Mouse RNA-Seq. Total RNA was extracted from intermolar eminence (IE) = tex], rabbit anti-Trp63 [GTX124660; Genetex], and rabbit anti-Bmi1 [RA25083; epithelium of E17.5 control and mutant embryos (n 3 of each) following Neuromics]) in conjunction with mouse IgG2a anti-BrDU (RPN202; GE Health- standard methods. In brief, tissue was homogenized with TRIzol (Invitrogen). care) and the provided blocking solution containing nuclease enzyme at 4 °C. RNA was separated with chloroform, precipitated with isopropanol, and Slides were then rinsed 2× 1 h in PBS and incubated in secondary antibodies at subsequently purified with RNeasy MINI kit (74104; Qiagen). RNA quantity 1:400 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Molecu- was measured, and quality was assessed with an Agilent Bioanalyzer (RNA > lar Probes) and Alexa Fluor 568 goat anti-mouse IgG2a (Molecular Probes) in 6000 nano; Agilent). One microgram of each high-quality (RIN 7) RNA blocking solution at room temperature. Unbound secondary antibody was sample was sent to the Oxford Genomics Centre for sequencing. Raw FASTQ removed by washing 2× 1 h in PBS, and the HRP signal was amplified using a files were trimmed based on End min quality level (Phred) and aligned BAM 488-tyramide chemistry signal amplification kit (Molecular Probes). Slides were files by BWA 0.7.12 to the mouse mm10 reference genome using Partek Flow again rinsed 2× 1 h and mounted with a 50:50 glycerin:Vectashield mixture for software with default parameters. Aligned reads were then quantified to the imaging using a Zeiss 710 confocal imaging system. annotation mode (mm10; Ensembl Transcripts release 83), and raw gene counts were normalized. Differential gene expression was identified by Partek Chemical Treatment. A 10-μm stock solution of LDN-193189 (LDN) (Enzo) was Gene-specific analysis. False discovery rate (FDR) correction with default P < prepared for each chemical treatment experiment using dimethyl sulfoxide value of 0.05 was considered statistically significant. (DMSO) (MP Biomedicals). All chemical and control experiments were performed in Erlenmeyer flasks at 28 °C in an oscillating platform culture incubator Mouse Whole Mount In Situ Hybridization. Freshly dissected tongues from −/− +/+ (Lab-Line Max 4000; Barnstead). For changes in gene expression assayed by ISH, Follistatin homozygous (Fst ) and wild-type mice (Fst ) at E17.5 were cichlids were raised to 20 dpf, and embryos from single broods were split into a fixed in 4% PFA overnight at 4 °C, followed by dehydration procedures small molecule treatment and a solvent control group. Treatments were per- through a methanol series (25%, 50%, 75%, and 100% methanol, 15 min per

formed at 4 μM LDN in 200 mL of fish H2O. After 48-h treatment in the small step). Samples were then rehydrated in PBS and bleached in 6% hydrogen molecule dilution, fry were killed immediately and fixed in 4% PFA. ISH was peroxide at room temperature for 1 h prior to proteinase K incubation and then carried out to assay effects of treatment on gene expression. washed with glycine in PBS and tween (PBT). Samples were refixed in 0.2% glutaraldehyde and 4% PFA in PBT before hybridization with DIG-labeled RNA Cichlid RNA Extraction and Sequencing. Animals (∼1-y-old adult MZ and LF probes at 70 °C overnight. After intensive posthybridization wash in solutions males) were killed and immediately dissected for RNA extraction. A ribbon containing 50% formamide and RNase treatment, followed by antibody ∼1mm× 10 mm of epithelium was removed labial to the outer row of teeth binding (anti–Digoxigenin-AP, 1:3,000; Roche) at 4 °C overnight, antibodies from the dentary of experimental animals using a no. 12 scalpel blade. The were stained with Alkaline Phosphate and BM-Purple (11442074001; Roche) extraosseous soft tissue was removed from the entire jaw to reduce the risk and were used for colorimetric detection. Samples were then photographed of TBs containing epithelium carryover. The bone was then shaved down using a Leica MZ FLIII microscope with Leica DFC300FX camera and Leica using a scalpel to expose the bony crypts, and intraosseous RT were FireCam software and transmitted lateral and or/direct illumination. extracted with fine forceps. Extracted tissue was quickly placed in RNAlater − − RNA Stabilization Reagent (Qiagen). Tissues were frozen in liquid nitrogen, Mouse Tissue Real-Time PCR. Mouse tongues were collect at E17.5 from Fst / and + + homogenized using a mortar and pestle, and placed in TRIzol. Following Fst / mice, and posterior intermolar eminence (IE) regions were carefully

Bloomquist et al. PNAS | September 3, 2019 | vol. 116 | no. 36 | 17865 Downloaded by guest on September 28, 2021 dissected in cold PBS. Dissected tissues were incubated with Dispase II the experiments are listed in Dataset S3. Ct values were normalized with β-actin −ΔΔ (04942078001; Roche) at 37 °C for 30 min before mechanically dissociating epi- levels as internal controls, and data were analyzed by 2 ct methods. Error bars thelium from underneath mesenchyme tissue. Dissociated epithelial tissues were are presented by SDs, which were calculated from biological triplicate samples. snap frozen by liquid nitrogen and stored at −80 °C prior to total RNA extraction. Total RNA was extracted from mouse tongue epithelia on IE regions using ACKNOWLEDGMENTS. We thank members of the J.T.S. laboratory and P.T.S. RNeasy Mini Kit (Qiagen; 74104) and purified by Ambion DNA-free DNA Re- laboratory for comments on previous drafts of this manuscript. This work moval Kit (AM1906; Invitrogen,). cDNA was then synthesized as described before was supported by National Institute of Dental and Craniofacial Research (15, 67). One microgram of cDNA was used for qPCR reaction applied on a (NIDCR) Grant R01 DE019637 (to J.T.S.) and National Institutes of Health LightCycler 480 system qPCR platform (05015278001; Roche). All primers used in (NIH) Grant 1F30 DE023013 (to R.F.B.).

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17866 | www.pnas.org/cgi/doi/10.1073/pnas.1821202116 Bloomquist et al. Downloaded by guest on September 28, 2021